Therapeutic proteins including peptide hormones, cytokines, and monoclonal antibodies have achieved widespread success as research tools and are among the fastest growing classes of drugs. Many powerful and potentially therapeutic proteins have been discovered or engineered over the past two decades, including enzymes capable of metabolic complementation (Hartung, S. D. et al. Gene. Mol. Ther. 9, 866-875 (2004)), neutralizing antibodies against intracellular targets (Wang, J. et al. Nat. Biotechnol. 26, 901-908 (2008)), engineered transcription factors (Urnov, F. D., et al. Nat. Rev. Genet. 11, 636-646 (2010)), and programmable genome-editing enzymes (Sander, J. D. & Joung, J. K. Nat. Biotechnol. 32, 347-355 (2014); Gaj, T., et al. Trends Biotechnol. 31, 397-405 (2013)). While protein biologics have proven effective for extracellular targets, their use to address intracellular targets is comparatively undeveloped due to the inability of most proteins to spontaneously enter mammalian cells. Enabling exogenous proteins to access intracellular targets is most commonly achieved by delivery of their encoding DNA sequences through chemical transfection (Midoux, P., et al. Br. J. Pharmacol. 157, 166-178 (2009)), electroporation (Bodles-Brakhop, A. M., et al. Mol. Ther. 17, 585-592 (2009)), or viral delivery (Kay, M. A., et al. Nat. Med. 7, 33-40 (2001)). The introduction of exogenous DNA into cells, however, raises the possibility of permanent recombination into the genome, potential disruption of endogenous genes, and long-term exposure to the encoded agent. For some research or therapeutic applications, including genome editing applications that seek to effect a one-time, permanent modification of genomic DNA, the functional delivery of non-replicable protein agents may offer improved safety or broader applicability.
The recent development of methods to deliver in vitro transcribed mRNAs or mRNA analogs has offered an alternative to DNA delivery without requiring nuclear transport of an encoding gene, and with greatly reduced potential for genomic insertion of the foreign nucleic acid. While promising, mRNA delivery continues to face challenges including immunogenicity and RNA stability. While chemical modifications and the inclusion of base analogs can mitigate some of these issues, the large-scale production of high-quality modified mRNAs remains a challenge (Zangi, L. et al. Nat. Biotechnol. 31, 898-907 (2013)). Moreover, proteins containing important natural or synthetic post-translational modifications may not be amenable to production by endogenous translation machinery. Therefore, while both DNA and mRNA delivery have become powerful research tools with therapeutic implications, the development of effective and general protein delivery methods remains an important challenge for the molecular life sciences.
Current or conventional protein delivery technologies are based on fusion or conjugation to cationic molecules that facilitate endocytosis, such as unstructured peptides (Wadia, J. S., et al. Nat. Med. 10, 310-315 (2004); Daniels, D. S. & Schepartz, A. J. Am. Chem. Soc. 129, 14578-14579 (2007)) or engineered superpositively charged proteins (Cronican, J. J. et al. ACS Chem. Biol. 5, 747-752 (2010); Thompson, D. B., et al. Methods Enzymol. 503, 293-319 (2012); Thompson, D. B., et al. Chem. Biol. 19, 831-843 (2012)). While such delivery can be effective in cell culture, and has even shown some success in vivo, cationic protein-based delivery methods have not seen widespread adoption. Unprotected proteins can be rapidly degraded by extracellular and endosomal proteases (Heitz, F., et al. Br. J. Pharmacol. 157, 195-206 (2009)), or neutralized by binding to serum proteins, blood cells, and the extracellular matrix (Caron, N. J. et al. Mol. Ther. J. Am. Soc. Gene Ther. 3, 310-318 (2001); Chesnoy, S. & Huang, L. Annu. Rev. Biophys. Biomol. Struct. 29, 27-47 (2000)). In addition, the low efficiency of endosomal escape and avoidance of lysosomal degradation are major challenges to all endocytic protein delivery strategies, as evidenced by ongoing interest in endosome altering (Thompson, D. B., et al. Chem. Biol. 19, 831-843 (2012); Al-Taei, S. et al. Bioconjug. Chem. 17, 90-100 (2006)) and destabilizing strategies (Shete, H. K., J. Nanosci. Nanotechnol. 14, 460-174 (2014)). These challenges have proven especially difficult in vivo (Aguilera, T. A., et al. Integr. Biol. Quant. Biosci. Nano Macro 1, 371-381 (2009)).
Nucleic acid delivery has benefited greatly from the development of liposomal reagents over the past two decades. Cationic lipid formulations have enabled DNA and RNA transfection to become a routine technique in basic research and have even been used in clinical trials (Coelho, T. et al. N. Engl. J. Med. 369, 819-829 (2013)). The lipid bilayer of the vehicle protects encapsulated nucleic acids from degradation and can prevent neutralization by antibodies (Judge, A. D., et al. Mol. Ther. J. Am. Soc. Gene Ther. 13, 494-505 (2006)). Importantly, fusion of liposomes with the endosomal membrane during endosome maturation can enable the efficient endosomal escape of cationic lipid-delivered cargo (Basha, G. et al. Mol. Ther. J. Am. Soc. Gene Ther. 19, 2186-2200 (2011)). More advanced reversibly ionizable lipid nanoparticles enable efficient encapsulation and delivery of nucleic acids, while avoiding non-specific electrostatic interactions and sequestration (Semple, S. C. et al. Nat. Biotechnol. 28, 172-176 (2010)).
Because proteins, in contrast to nucleic acids, are chemically diverse with no dominant electrostatic property, no lipid formulation is likely to drive the efficient delivery of all proteins into mammalian cells. While proteins can be encapsulated non-specifically and delivered by rehydrated lipids in vitro (Boeckle, S., et al. J. Control. Release Off. J. Control. Release Soc. 112, 240-248 (2006); Allen, T. M. & Cullis, P. R. Adv. Drug Deliv. Rev. 65, 36-48 (2013)), the efficacy of encapsulation is dependent on protein concentration, is generally inefficient (Zelphati, O. et al. J. Biol. Chem. 276, 35103-35110 (2001)), and has not seen widespread application. Specialty commercial reagents developed specifically for protein delivery (Adrian, J. E. et al. J. Control. Release Off. J. Control. Release Soc. 144, 341-349 (2010); Morris, M. C., et al. Nat. Biotechnol. 19, 1173-1176 (2001)) have also failed to garner popularity perhaps due to their low potency and unreliability with a variety protein cargoes (Colletier, J.-P., et al. BMC Biotechnol. 2, 9 (2002)).